Testosterone of formula (I):
also named (17β)-17-hydroxyandrost-4-en-3-one, is a steroid, a known human hormone and an active ingredient largely employed for pharmaceutical purposes.
It is well known that for hydrolyzing esters compounds, basic medium can be successful employed, in particular, aqueous soda is a typical reagent. Therefore, it is well known to the skilled person aimed to prepare testosterone that aqueous caustic soda can be used to convert by hydrolysis testosterone esters into testosterone.
Nevertheless, experimental attempts to convert testosterone esters to testosterone by using aqueous caustic soda provides testosterone having a large amounts of several unknown impurities, which are very difficult to be eliminated from the product, for example through re-crystallization procedures.
Several other methods for the preparation of testosterone have been disclosed. In particular, some of them describe the transformation of said steroid by means of enzymatic hydrolysis.
Specifically, Tetrahedron Letters, 28 (52), 6549-6552, 1987 discloses a procedure for the conversion of steroid esters into the corresponding alcohols, i.e. steroids, via enzymatic transesterification in organic solvent. Said enzymatic transformation, after an initial screening of a number of hydrolytic enzymes, revealed that lipase from Candida cylyndracea was found to be the best catalyst to hydrolyse steroid esters with octanol in organic solvents. The results of some reactions are shown in the table of said article at pages 6551-6552, in particular testosterone esters substrates were tested with said lipase catalyst (see entry 9 and 10). The results show how the lipase from Candida cylyndracea did not produce any conversion of testosterone acetate to testosterone, as well as the same reaction with testosterone propionate gives 12% of conversion, only. Furthermore, this enzymatic reaction was carried out in organic solvents with a high amount of enzyme and a low amount of substrate.
The publication Steroids, 62, 482-486, 1997 discloses a direct stereoselective synthesis of a testosterone derivative, being the 7β-hydroxytestosterone (9) by enzymatic oxidation followed by an enzymatic hydrolysis. In particular, said method describes the use of porcine lipase to obtain the compound (9), from the intermediate ester. This enzymatic hydrolysis using said lipase, from porcine pancreas, produces a conversion of 7β,17β-dihydroxy-4-androsten-3-one 17-caprylate (8) to 7β-hydroxytestosterone (9). The H-NMR spectrum of the compound (9) shows the absence of the ester group. At the same time, said conversion results in a 75% yield but the product needed to be purified by flash column chromatography. Therefore said method is not suitable for large productions. Moreover, it is observed that the product obtained by such enzymatic hydrolysis, is the compound (9), which is 7β-hydroxytestosterone, i.e. a different compound than testosterone.
Finally, Acta Chemica Scandinavica 27, 1240-1248, 1973 discloses a transformation of steroids by cell-free preparation of Penicillium lilacinum NRRL 895; the latter shows to contain inducible steroid esterase activity. The article describes the steroid transforming capacity of the fungus Penicillium lilacinum that was tested on several steroidal compounds, in particular, on testosterone and esters thereof.
In the specific case, in two experiments testosterone acetate was readily hydrolysed to testosterone with conversions of 80.3% and 83.8% (see pag. 1246). Nevertheless, the use of such esterase from said cell-free on other testosterone esters like testosterone propionate provides only partial hydrolysis, whereas testosterone enanthate, benzoate and hemisuccinate were unaffected. Moreover, it should be taken in account that these results were obtained, and in particular the transformation of testosterone acetate to testosterone, at very low concentration of the substrate, were unsuitable for commercial and/or industrial productions.